A High Density Distributed Three-dimensional Electrode Device

ABSTRACT

A high-density distributed three-dimensional electrode device, comprising an electrode array and an electrode fixing assembly on which the electrode array is fixed, the electrode array comprising a plurality of electrodes respectively applied with an electric pulse of a first polarity and an electric pulse of a second polarity according to a time period, wherein the electrodes corresponding to the electric pulse of the second polarity are distributed around the electrodes corresponding to the electric pulse of the first polarity.

TECHNICAL FIELD

The present invention relates to a cell electroporation technique, inparticular a three-dimensional electrode device for electroporation.

BACKGROUND

Since 1970s, the electroporation technique was used to insert moleculesinto animal cells or plant cells. It is proved by researchers thatexposing a cell to a short-lasted high-voltage electric field may enableformation of pathways through the cell membrane, and macromolecules suchas proteins and DNAs may enter into the cell through those pathways.Those pathways are reffered as electric pores which are permeabilityincreased zone caused by a local fracture of cell membrance resultedfrom high voltage electric field. Although the existing times of thepores are brief, it is enough to satisfy the requirement of themacromolecules such as plasmid DNA molecules entering into the cell. Thecell may tolerate the formation of the pores, however, the cell may bekilled by the processes of the formation and the molecules introducedthereby if the formed pores are too much and overlarge.

At the earliest, the electroporation is carried out by using thesimplest capacitor with parallel-plate, and a substantially homogeneouselectric field may be formed between the electrodes opposite to eachother. The cell suspension prepared for electroporation and themolecules which the operator wants to introduce are mixed and placedbetween the two electrodes, and a short-time high voltage electric fieldpulse is applied to the electrodes by one or more times such that theresult of introducing the molecules into cells by electroporation can beachieved. However, the distance between the parallel-plate electrodes islarge, the required voltage is usually up to several thousands volts,thus generating of cathode effect is inevasible, which has a huge damageto the cells.

Although the planar electrodes arose later solve the negative effectbrought by the overhigh voltage, they are not suitable for highthroughput experiment operations due to that the planer electrodes canprocess a very small amount of cells every time. Three-dimensionalelectrodes easily penetrate into tissues and living bodies, and usuallyuse for electroporations in clinic for tumour tissues or living tissues,the electroporation efficiency of which is not high, and there is norelated report of extracorporeal cell electroporation such aselectroporation aimed at suspended cells or attached cells viathree-dimensional electrodes.

SUMMARY

The present invention provides a high-density distributedthree-dimensional electrode device, which has a simple structure and iseasy to manufacture.

To solve the above-mentioned technical problems, the present inventionprovides a high-density distributed three-dimensional electrode device,comprising an electrode array and an electrode fixing assembly on whichthe electrode array is fixed, the electrode array comprising a pluralityof electrodes divided into at least two groups, an electric pulse of afirst polarity and an electric pulse of a second polarity arerespectively applied on the at least two groups according to a timeperiod, wherein the first polarity and the second polarity aredifferent, and the electrodes corresponding to the electric pulse of thesecond polarity are distributed around the electrodes corresponding tothe electric pulse of the first polarity.

In a preferred embodiment of the present invention, the plurality ofelectrodes in the electrode array is arranged according to anequilateral polygon, and the distances between every two adjacentelectrodes in the electrode array are equal.

In a preferred embodiment of the present invention, a shape of theelectrode array is an equilateral hexagon formed by several equilateraltriangles, and the electrodes are located at the vertexes of theequilateral triangles.

In a preferred embodiment of the present invention, the first polarityis positive polarity and the second polarity is negative polarity.

In a preferred embodiment of the present invention, the first polarityis negative polarity, and the second polarity is positive polarity.

In a preferred embodiment of the present invention, the diameters of theelectrodes are 0.01-1.2 mm, the distance between the center points ofthe two adjacent electrodes is 0.1-2.4 mm, the number of the electrodesis more than 5, and the number is preferred to be more than 19. Thematerial of the electrodes is preferably stainless steel.

In a more preferred embodiment of the present invention, the diametersof the electrodes are 0.1-0.4 mm, the distance between the center pointsof the two adjacent electrodes is 0.2-1.5 mm, and the number of theelectrodes is more than 36. The diameters of the electrodes arepreferably 0.3 mm, the distance between the center points of the twoadjacent electrodes is preferably 1 mm, and the number of the electrodesis preferably 37.

In a preferred embodiment of the present invention, the electrode fixingassembly comprises an electrode connecting circuit board and anelectrode positioning board, the electrode connecting circuit boardconnecting the electrodes applied with the electric pulse of the samepolarity together via a line thereon, and the electrodes being insertedinto the electrode positioning board.

In a preferred embodiment of the present invention, the electrodes inthe electrode array are divided into several groups, and the electrodesin the same group are only applied with the electric pulse of the samepolarity, wherein one of the groups is applied with the electric pulseas a positive electrode, the rest groups are applied with the electricpulse as a negative electrode, and then another one of the groups isapplied with the electric pulse as the positive electrode, the restgroups are applied with the electric pulse as the negative electrode,and alternately in this way, the obtained electric field may achieve ahomogeneous electric field by superposition.

The beneficial effects of the present invention are that: the highdensity distributed three-dimentional electrode device of the presentinvention employs grouped reused electrodes; can compensate for theunevenness of the electric field caused by the three-dimentionalelectrode at the greatest extent; can process milliliter level of cellsonce; can be used in both pore plate devices and flow devices; requiresa small electroporation voltage due to a small distance between theelectrodes, avoiding the damage to the cells from the high voltage; lowcost; is a cell electroporation device with high throughput and highefficiency.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a structure schematic diagram of a preferable embodiment of ahigh-density distributed three-dimentional electrode device according tothe present invention.

FIG. 2 is a front view of the high-density distributed three-dimentionalelectrode device shown in FIG. 1.

FIG. 3 is a top view of the high-density distributed three-dimentionalelectrode device shown in FIG. 1.

FIG. 4 is a topological structure diagram of an electrode array in thehigh-density distributed three-dimentional electrode device shown inFIG. 1.

Wherein reference numbers and corresponding parts are as follow:1.electrode array, 2.electrode positioning plate, 3.electrode connectingcircuit board.

DETAIL DESCRIPTION OF EMBODIMENTS

In the following, the preferable embodiments of the present inventionare explained in detail combining with the accompanying drawings so thatthe advantages and features of the present invention can be easilyunderstood by the skilled persons in the art, and thus it is clear todefine the protective scope of the present invention.

Embodiment 1

Referring to FIG. 1, FIG. 2, FIG. 3 and FIG. 4, the present inventionprovides a high-density distributed electrode device, comprising anelectrode array 1 and an electrode fixing assembly on which theelectrode array 1 is fixed.

The electrode array 1 is formed by 37 solid cylinder electrodes, and thearrangement rules are as follow: all the electrodes are arranged to bean equilateral hexagon shaped structure, the distances between twoadjacent electrodes are equal, the inside of the equilateral hexagonshape is divided into several small equilateral triangle units by takingthe distances between two adjacent electrodes as side length, and oneelectrode is placed at each vertex of the equilateral triangle units,that is, all the electrodes are divided into three groups, Group I, IIand III, and vertexes of each equilateral triangle unit respectivelybelong to Group I, II and III.

The inner diameter of the equilateral hexagon of the electrode array 1is matched up with the plate holes of a perforated plate, and a distancebetween the center points of two adjacent electrodes is 1 mm. Thedistance between two electrodes may affect the voltage duringelectroporation, and can be adjusted as required. The electrodes areinserted into the perforated plate and have a distance of 0.1 mm-1 mmfrom the bottom of the perforated plate.

The diameter of the electrodes is 0.3 mm, and both too large and toosmall diameters of the electrodes may affect the effect of theelectroporation. When the diameters are too large, the effective area ofelectric field may be reduced, resulting in a decrease on the number ofcells dealed with by electroporation, and going against highthroughputof cell electroporation. When the diameters are too small, theelectrodes are easily bended resulting in a large increase of themanufacturing cost.

The material of the electrodes may be optionally selected from electricconductive metals and other electric conductive materials, wherein thestainless steel is an excellent material for electrodes. The stain steelmaterial possesses favorable bio-compatibility, is easy to clean and noteasy to be oxidized, easy to form relatively long electrodes, and isable to be mass produced and be reused for multiple times withoutaffecting the conduction properties thereof.

The electrode fixing assembly comprises an electrode positioning board 2and an electrode connecting circuit board 3, the electrodes arepositioned by going through the electrode positioning board 2 andconnected to the electrode connecting circuit board 3, and the electrodeconnecting circuit board 3 connects the electrodes applied with theelectric pulse of the same polarity together via a line thereon. Due tothat the electrodes are relatively long and the electroporation has avery high accuracy requirement for the distance between the electrodes,the electrode positioning board 2 is employed to position the electrodesin electroporation experiments. The electrode positioning board 2 canconfine and position the electrodes along a long distance due to athickness thereof is about 1 cm, and therefore, the electrodes can becontrolled accurately to reach the bottom of the perforated plate. Asupport structure of the high density distributed three-dimentionalelectrode device may be easily extended to form electrode networks ofany combination array such as 2*2, 1*4, 12*8 etc., and such flexible andvaried conbinations may be compatible with the perforated platestructure at the greatest extent, being conveniet for user.

The electrode connecting circuit board 3 connects the electrodes in thesame group together by welding connection, conducting adhesive or otherelectrical connecting manner such as printed circuit board and anyothercomponet which can connect regulation lines. The electrode positioningboard 2 is used for positioning the electrodes which are vimineous andeasily bended. Therefore, in the embodiments of FIGS. 1-2, the electrodepositioning board 2 is arranged approximately at the middle of theelectrodes for positioning. The use of the electrode positioning board 2can reduce the inconformity of the distances between the electrodes andthus improve the homogeneity of the electric field.

The high-density distributed three-dimensional electrode device may be amonoporate device, and 4, 96 or more of this device can be used to forma group to cooperate with a perforated plate structure commonly used inbiology.

The electroporation method of the high-density distributedthree-dimensional electrode device in the present embodiment is: duringthe electroporation, firstly by taking Group I as a positive electrodeand Group II, III as a negative electrode the electric pulse is applied,then by taking Group II as a positive electrode and Group I, III as anegative electrode the electric pulse is applied, and then by takingGroup III as a positive electrode and Group I, II as a negativeelectrode the electric pulse is applied.

For certain kind of cells, pores may appear on the cell membranes whenthe electric field is higher than a certain threshold. The death rate ofthe cells may arise as the electric field gradually increases. To ensurea high electroporation rate and low death rate of the cells, it isdesired to accurately control the electric field to be the thresholdelectric field of the electroporation.

As long as the homogeneous electric field intensity is controlled to bethe optimal electroporation voltage of the electroporation, cells in thewhole effective area may experience electroporation at the greatestextent. It can be seen that grouping and reusing compensate for theunevenness of the electric field caused by single group, increases theelectroporation efficiency, and thus it can be determined that such kindof combination of electric fields has a much higher electroporationefficiency than the conventional electroporation.

The high-density distributed three-dimensional electrode device maycarry out electroporation for many cell lines in suspension or inadherence. 12 kinds of cells, 7 HEK-293A, Hela, MCF-7, A-375, Neuro-2A,U251, C2C12, 3T3-L1, CHO, MDCK, HL-60, HUVEC, are choosen to undergoelectroporation, and GFP molecules are used as marker. The GFP moleculesmay enter into the cells and synthesize fluorescent substances in thecells if the cells are electroporated, and the synthesized fluorescentsubstances may glow green fluorescence under a fluorescent field suchthat the electroporation rate of the cells may be obtained from thenumber of cells in the fluorescent field divided by the total number ofcells, that is to say, the higher the fluorescence intensity of the samedensity of cells is, the higher the efficiency of the electroporationis.

The high-density distributed three-dimensional electrode deviceaccording to the present invention may be applied in a flow device. Byplacing the high-density distributed three-dimensional electrode devicein the flow device, the cells evenly distributed and flowing along withthe flowing fluid may accept an optimal electroporation stimulationconditions when the cells experience the control of the flowing rate andpulse stimulation during passing through the electrode array, theelectroporation stimulation conditions comprising the voltage amplitudevalue of pulse, pulse width, pulse interval, pulse number and electrodeswapping control. In a continuous flowing system, except that the timewhen the pulses are applied to the electrodes and the time when thecells begin to flow among the electrodes need to be harmonized, thepulses and the flowing of the cell do not need a harmonization of“duration handling” between them.

The high-density distributed three-dimensional electrode deviceaccording to the present invention both has a advantage of continuousflow electroporation, in particularly being able to carry out ahigh-throughput electroporation for cells in a sterile closed system,and is also able to ensure that each of the cells is subjected withoptimal number of pulses and most even electric field to improve theelectroporation efficiency and low down the death rate.

Embodiment 2

The high-density distributed three-dimensional electrode deviceaccording to the present embodiment is similar to that of Emobodiment 1differing in the arrangement of the electrode array. In the presentembodiment, the arrangement rule of the electrode array is as follow:all the electrodes are arranged to form an equilateral quadrangle shapedstructure, the distances between the adjacent two electrodes are equal,the inside of the equilateral quadrangle is divided into several smallersquare units by taking the distances between the adjacent two electrodesas side length, and one electrode is placed at each vertex of thesquares, i.e. all the electrodes are divided into four groups, Group I,II, III, and IV, and vertexes of each square respectively belong to

Group I, II, III, and IV, and these groups take turns to be applied withthe electric pulse by taking one of the groups as a positive electrodeand the rest groups as a negative electrode. The arrangement of theelectrode array of this embodiment may be represented by slightlymodifying the circumstances of Embodiment 1 shown in FIG. 4, andtherefore no more figures are shown.

Embodiment 3

The high-density distributed three-dimensional electrode device providedby the present embodiment is similar to that of Emobodiment 1 differingin that the arrangement of the electrode array is as follow: all theelectrodes are arranged to form an equilateral hexagon structure, thedistances between the adjacent two electrodes are equal, the inside ofthe equilateral hexagon is divided into several smaller equilateralhexagon units by taking the distances between the adjacent twoelectrodes as side length, and one electrode is placed at each vertex ofthe hexagon units, i.e. all the electrodes are divided into six groups,Group I, II, III, IV, V, and VI, and vertexes of each hexagon unitrespectively belong to Group I, II, III, IV, V, and VI, and these groupstake turns to be applied with the electric pulse by taking one of thegroups as a positive electrode and the rest groups as a negativeelectrode. The arrangement of the electrode array of the presentembodiment may be represented by slightly modifying the circumstances ofEmbodiment 1 shown in FIG. 4, and therefore no more figures are shown.

The above are only embodiments of the present invention, and are no wayto limit the scope of the present invention. Any equivalent structuresor process changes, or direct or indirect application on other relativetechnical fields by taking advantage of the content of the presentinvention should be covered by the scope of the present invention.

1. A high-density distributed three-dimensional electrode devicecharacterized in that: the device comprises an electrode array and anelectrode fixing assembly on which the electrode array is fixed, theelectrode array comprising a plurality of electrodes divided into atleast two groups, an electric pulse of a first polarity and an electricpulse of a second polarity are respectively applied on the at least twogroups according to a time period, wherein the electrodes correspondingto the electric pulse of the second polarity are distributed around theelectrodes corresponding to the electric pulse of the first polarity,and the first polarity and the second polarity are opposite.
 2. Thedevice as claimed in claim 1, wherein the diameters of the electrodesare 0.01-1.2 mm, the distance between the center points of two adjacentelectrodes is 0.1-2.4 mm, and the number of the electrodes is more than5.
 3. The device as claimed in claim 2, wherein the diameters of theelectrodes are 0.1-0.4 mm, the distance between the center points of twoadjacent electrodes is 0.2-1.5 mm, and the number of the electrodes ismore than
 36. 4. The device as claimed in claim 3, wherein the diametersof the electrodes are 0.3 mm, the distance between the center points oftwo adjacent electrodes is 1 mm, and the number of the electrodes is 37.5. The device as claimed in claim 1, wherein the plurality of electrodesin the electrode array is arranged according to an equilateral polygon,and the distances between every two adjacent electrodes in the electrodearray are equal.
 6. The device as claimed in claim 5, wherein a shape ofthe electrode array is an equilateral hexagon formed by severalequilateral triangles, and the electrodes are located at the vertexes ofthe equilateral triangles.
 7. The device as claimed in claim 1, whereinthe material of the electrodes is stainless steel.
 8. The device asclaimed in claim 1, wherein the electrode fixing assembly comprises anelectrode connecting circuit board and an electrode positioning board,and the electrode connecting circuit board connects the electrodesapplied with the same electric pulse together, the electrodes areinserted into the electrode positioning board.
 9. The device as claimedin claim 1, wherein the electrodes in the electrode array are dividedinto three or more groups, the electrodes in the same group are appliedwith the electric pulse of the same polarity, and the groups ofelectrodes are applied with an electric pulse according to a time periodto achieve a homogeneous electric field by superposition of the resultedelectric fields in the following alternate way: one of the groups isapplied with the electric pulse as a positive electrode, the rest groupsare applied with the electric pulse as a negative electrode, and thenanother one of the groups is applied with the electric pulse as thepositive electrode, the rest groups are applied with the electric pulseas the negative electrode.